Tumor hypoxia-responsive nitric oxide prodrug compounds, methods of making and using the same

Abstract

The present application provides a tumor hypoxia-responsive nitric oxide prodrug compound, which can not only realize accurate activation by overexpressing reductase in the hypoxic tumor microenvironment, release NO donors and play an anti-tumor role, but also realize accurate positioning and visual diagnosis of tumors by releasing beta-carboline fluorophore. The design of this diagnosis and treatment integration overcomes the limitations of traditional chemotherapy methods, provides a new solution for precise tumor treatment, and has important clinical transformation value and broad application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a tumor hypoxia-responsive nitric oxide therapeutic agent, its preparation method, and its application. Background Technology

[0002] Malignant tumors are one of the leading causes of death worldwide, and their continuously rising incidence and mortality rates make the development of novel cancer treatment strategies particularly important. The tumor microenvironment (TME) plays a crucial role in the occurrence, development, and metastasis of tumors, with the hypoxic microenvironment of tumor tissue being a prominent feature. Under hypoxic conditions, the expression levels of various biological reductases in tumor cells, such as nitroreductase (NTR) and azoreductase (AzoR), are significantly increased. The expression level of AzoR is closely related to the degree of hypoxia in tumor tissue, providing a theoretical basis for the development of prodrugs based on AzoR responses. In 2017, Kang et al. used azobenzene as a linker to connect the fluorophore rhodamine 123 / B to nitrogen mustard, successfully achieving the effect of AzoR-activated drugs in hypoxic tumors. When the drug is activated by AzoR in hypoxic tumors, nitrogen mustard is released to exert its anticancer effect, and the fluorescent chemical rhodamine, as a diagnostic tool, provides important visual support for tumor localization and diagnosis. This study provides an important reference for AzoR-based prodrug design and demonstrates the potential for precision diagnosis and treatment in the tumor microenvironment.

[0003] However, traditional chemotherapy methods, due to their systemic effects, are often accompanied by significant toxic side effects, limiting the clinical application of these drugs. Triggered drug activation, as a promising strategy, utilizes the tumor microenvironment or external stimuli to activate prodrugs, releasing active ingredients and thereby improving treatment efficacy while reducing damage to normal tissues. This strategy not only improves treatment precision but also reduces potential damage to healthy cells. The new medical model of integrated diagnosis and treatment further integrates diagnostic and therapeutic functions, enabling real-time acquisition of in vivo information during treatment and rational control of drug dosage, thus reducing toxic side effects. This model not only improves treatment efficiency but also reduces patient suffering and risks, becoming an important development direction in the field of cancer treatment.

[0004] β-Carboline, a natural indole alkaloid, possesses a unique planar conjugated structure and broad bioactivity, making it a promising candidate for fluorescence imaging. Recent studies have revealed that 3-amino-β-carboline exhibits fluorescence sensitivity under acidic pH conditions, further expanding its potential applications in diagnosis and treatment. Nitric oxide (NO) is a gaseous signaling molecule with diverse biological activities. At high concentrations, NO can activate cytotoxic defense mechanisms, participate in the metastasis and progression of various tumors, and exert its anti-tumor effects by regulating the expression of angiogenesis, cell cycle, and apoptosis-related proteins. Due to the short half-life of NO in vivo, the development of NO donor drugs has become a major research direction. Against this background, the development of a tumor hypoxia-responsive nitric oxide prodrug compound is of great significance. Summary of the Invention

[0005] The purpose of this invention is to overcome the deficiencies in the prior art and provide a tumor hypoxia-responsive nitric oxide prodrug compound.

[0006] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0007] A tumor hypoxia-responsive nitric oxide prodrug compound has the following structural formula:

[0008] ,

[0009] Wherein, R is selected from: , , , or Y is selected from Cl, Br, I, and PF6.

[0010] More specifically, the tumor hypoxia-responsive nitric oxide prodrug compound of the present invention is (Z)-1-((4-((E)-(1,9-dimethyl-3-((E)-2-(1-methylquinoline-1-iodonium-4-yl)vinyl)-9H-pyrido[3,4-b]indol-6-yl)diazeninyl)benzyl)oxy)-3,3-diethyltriaza-1-ene-2-oxide, (E)-1-((4-((E)-) -(1,9-dimethyl-3-((E)-2-(1-methylquinoline-1-iodonium-4-yl)vinyl)-9H-pyrido[3,4-b]indol-6-yl)diazenyl)benzyl)oxy)-3-(2-hydroxyethyl)-3-methyltriazol-1-en-2-oxide, (E)-2-((4-((E)-(1,9-dimethyl-3-((E)-2-(1-methylquinoline-1-iodonium-4-yl)vinyl)-9H-pyrido[3,4-b]indol-6-yl)diazenyl)benzyl)oxy)-1-(pyrrolidine-1-yl)diazen-1-oxide, (E )-2-((4-((E)-(1,9-dimethyl-3-((E)-2-(1-methylquinoline-1-iodon-4-yl)vinyl)-9H-pyrido[3,4-b]indol-6-yl)diazenyl)benzyl)oxy)-1-morpholinodiazen-1-oxide, or (E)-2-((4-((E)-(1,9-dimethyl-3-((E)-2-(1-methylquinoline-1-iodon-4-yl)vinyl)-9H-pyrido[3,4-b]indol-6-yl)diazenyl)benzyl)oxy)-1-(4-hydroxypiperidin-1-yl)diazen-1-oxide).

[0011] In some embodiments, R is preferably... The preferred prodrug compound for tumor hypoxia-responsive nitric oxide is (E)-2-((4-((E)-(1,9-dimethyl-3-((E)-2-(1-methylquinoline-1-iodon-4-yl)vinyl)-9H-pyrido[3,4-b]indol-6-yl)diazeninyl)benzyl)oxy)-1-morpholinodiazenin-1-oxide.

[0012] The present invention also provides a method for preparing the above-mentioned tumor hypoxia-responsive nitric oxide prodrug compound, comprising the following steps: reacting bromo-4-((E)-2-(6-((E)-(4-(bromomethyl)phenyl)diazeninyl)-1,9-dimethyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinolineonium salt (7) with azoonium oxide RNa to obtain the above-mentioned tumor hypoxia-responsive nitric oxide prodrug compound.

[0013] Among them, bromo-4-((E)-2-(6-((E)-(4-(bromomethyl)phenyl)diazeninyl)-1,9-dimethyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinolineonium salt (7) is prepared by the following method: (E)-4-(2-(1,9-dimethyl-6-nitro-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinolineonium salt (4) is reduced with iron powder to obtain (E)-4-(2-(6-amino-1, 9-Dimethyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinolineonium salt (5) is then reacted with 4-nitrosobenzyl alcohol under acetic acid catalysis to generate 4-((E)-2-(6-((E)-(4-(hydroxymethyl)phenyl)diazeninyl)-1,9-dimethyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinolineonium salt (6). Finally, the bromo-4-((E)-2-(6-((E)-(4-(bromomethyl)phenyl)diazeninyl)-1,9-dimethyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinolineonium salt (7) is obtained by the Appel reaction under the action of CBr4 and PPh3.

[0014] The complete synthesis route is shown below:

[0015]

[0016] The present invention also provides a pharmaceutical composition comprising the above-described tumor hypoxia-responsive nitric oxide prodrug compound and a pharmaceutically acceptable carrier or excipient.

[0017] The present invention also provides the application of the above-mentioned tumor hypoxia-responsive nitric oxide prodrug compound in the preparation of a fluorescent imaging reagent targeting liver tumors, and in the preparation of a drug targeting liver tumor localization.

[0018] This invention also provides the application of the above-mentioned tumor hypoxia-responsive nitric oxide prodrug compound in the preparation of tumor therapeutic drugs. More specifically, the application of the above-mentioned tumor hypoxia-responsive nitric oxide prodrug compound in the preparation of drugs for treating colon cancer and breast cancer.

[0019] The present invention has the following advantages over the prior art:

[0020] (1) The compound of the present invention integrates diagnostic and therapeutic functions, enabling precise diagnosis and treatment in the tumor microenvironment. Under normal physiological conditions, the azo group shuts down the fluorescent diagnostic and therapeutic activities, while in the hypoxic tumor microenvironment, after the azo group is degraded, it releases a β-carboline fluorophore and a NO donor, thereby achieving targeted diagnosis and efficient treatment of tumors.

[0021] (2) The compounds of the present invention can respond to the overexpression of AzoR in the hypoxic tumor microenvironment and selectively degrade azo groups at the tumor site, thereby releasing diagnostic and therapeutic components. This responsive release mechanism improves the specificity and therapeutic effect of the drug, reduces potential damage to normal tissues, and provides strong support for precise tumor localization and surgical resection.

[0022] (3) Compared with existing drugs (such as Capecitabine), the compounds of the present invention exhibit a strong inhibitory effect on the growth of colorectal tumors, showing their great potential in tumor treatment. Attached Figure Description

[0023] Figure 1 The chemical structural formulas of the compounds in the embodiments of this invention are shown below;

[0024] Figure 2 The fluorescence emission spectrum of the compound of the present invention in Example 6 in response to AzoR is shown.

[0025] Figure 3 The selectivity and toxicity of the compound of the present invention to tumor cells in Example 7;

[0026] Figure 4 This refers to the nitric oxide content detection test of the compound of the present invention in Example 8;

[0027] Figure 5 In vivo fluorescence imaging and distribution testing of the compound of the present invention in nude mice in Example 9;

[0028] Figure 6 This is a test of the antitumor effect of the compound of the present invention in nude mice in Example 10. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0030] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0031] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples are commercially available products.

[0033] Example 1

[0034] Preparation of (Z)-1-((4-((E)-(1,9-dimethyl-3-((E)-2-(1-methylquinoline-1-iodon-4-yl)vinyl)-9H-pyrido[3,4-b]indol-6-yl)diazeninyl)benzyl)oxy)-3,3-diethyltriaza-1-ene-2-oxide (compound I1):

[0035] The synthetic route of the compounds of this invention is as follows:

[0036] Compound 4 (500 mg, 1.0 mmol), iron powder (273.73 mg, 4.0 mmol), and ammonium chloride (522.92 mg, 8.0 mmol) were added to a single-necked flask, dissolved in anhydrous ethanol (20 ml), and refluxed at 80 °C for 5 h. After the reaction was completed by TLC monitoring, the mixture was filtered, the filtrate was evaporated to dryness, and purified by column chromatography to obtain compound 5 in a yield of 72.6%.

[0037] The spectral data for compound 5 are as follows: 1H NMR (400 MHz, DMSO-d6) δ 9.36 (d, J = 6.5 Hz,1H, ArH), 9.30 (d, J = 2.3 Hz, 1H, ArH), 8.92 (d, J = 8.1 Hz, 1H, ArH), 8.62(m, 1H, ArH), 8.49 (dd, J = 9.2, 2.4 Hz, 2H, 2ArH), 8.34 (m, 1H, ArH), 8.13(d, J = 7.6 Hz, 1H, ArH), 7.97 (s, 1H, ArH), 7.71 (dd, J = 7.7, 1.6 Hz, 1H,ArH), 7.48 (td, J = 7.6, 1.1 Hz, 1H, CH=C), 7.26 (m, 1H, CH=C), 4.57 (s, 3H,OCH3), 4.47 (s, 2H, CH2), 4.30 (s, 3H, CH3), 3.18 (d, J = 6.0 Hz, 3H, CH3).

[0038] The structure of compound 5 was determined to be: (E)-4-(2-(6-amino-1,9-dimethyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinoline-1-iodonium salt.

[0039] (4-Nitrophenyl)methanol (139.01 mg, 22.0 mmol) was added to a single-necked flask and dissolved in anhydrous ethanol (15 ml). Then, compound 5 (500 mg, 28.6 mmol) and CH3COOH (3 ml) were added. The mixture was stirred at room temperature for 8 h. After the reaction was completed by TLC monitoring, the reaction solution was added to ice water to precipitate the solid. The solid was filtered, the filter cake was dried under vacuum, and purified by column chromatography to obtain compound 6 in 78% yield.

[0040] The spectral data for compound 6 are as follows: 1H NMR (400 MHz, DMSO-d6) δ 9.37 (d, J = 6.5 Hz,1H, ArH), 8.93 (m, 2H, 2ArH), 8.84 (m, 1H, ArH), 8.59 (dd, J = 9.2 Hz, 1H,ArH), 8.53 (m, 1H, ArH), 8.47 (m, 1H, ArH), 8.32 (m, 1H, ArH), 8.27 (m, 1H,ArH), 8.23 ​​(m, 1H, ArH), 8.18 (m, 2H, 2ArH), 8.12 (d, J = 7.6 Hz, 1H, ArH), 8.01 (m, 2H, ArH, CH=C), 7.95 (m, 1H, CH=C), 4.54 (s, 3H, CH3), 4.27 (s, 3H,CH3), 3.92 (s, 2H, CH2), 3.15 (s, 3H, CH3).

[0041] The structure of compound 6 was identified as: 4-((E)-2-(6-((E)-(4-(hydroxymethyl)phenyl)dienyl)-1,9-dimethyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinoline-1-iodonium salt.

[0042] Compound 6 (360 mg, 0.8 mmol), triphenylphosphine (236.72 mg, 1.0 mmol), and carbon tetrabromide (295.95 mg, 1.0 mmol) were added to a single-necked flask, dissolved in anhydrous THF (10 ml), and stirred at room temperature for 1 h. After the reaction was completed by TLC monitoring, the mixture was extracted and concentrated to give bromo-containing compound 7 in 85% yield.

[0043] Sodium (Z)-3,3-diethyltriaza-1-ene-2-oxide (200 mg, 1.2 mmol) was dissolved in THF (3 ml) at 0 °C under nitrogen protection. A solution of compound 7 (379.30 mg, 1 mmol) in DMF (1 ml) was added to the reaction mixture, and the mixture was stirred at room temperature for 4 hours. After the reaction was completed by TLC monitoring, the mixture was extracted with DCM, concentrated, and purified by column chromatography to obtain compound I1 in 75% yield.

[0044] The spectral data of compound I1 are as follows: 1H NMR (400 MHz, DMSO-d6) δ 9.34 (d, J = 6.5 Hz,1H, ArH), 8.91 (m, 2H, 2ArH), 8.84 (m, 1H, ArH), 8.53-8.58 (m, 2H, ArH), 8.46(m, 1H, ArH), 8.31 (m, 1H, ArH), 8.25 (m, 1H, ArH), 8.18-8.21 (m, 3H, 2ArH), 8.11 (d, J = 7.6 Hz, 1H, ArH), 8.01 (m, 2H, ArH, CH=), 7.94 (m, 1H, CH=), 4.76 (s, 2H, CH2), 4.54 (s, 3H, CH3), 4.26 (s, 3H, CH3), 3.15 (s, 3H, CH3), 2.59 (m, 4H, 2CH2), 1.02-1.06 (m, 6H, 2CH3).

[0045] The structural formula of compound I1 was identified as follows: Figure 1 As shown.

[0046] Example 2

[0047] Preparation of (E)-1-((4-((E)-(1,9-dimethyl-3-((E)-2-(1-methylquinoline-1-iodon-4-yl)vinyl)-9H-pyrido[3,4-b]indol-6-yl)diazenyl)benzyl)oxy)-3-(2-hydroxyethyl)-3-methyltriazol-1-en-2-oxide (compound I2):

[0048] Referring to the synthesis method of compound I1 in Example 1, (E)-1-onium salt-3-(2-hydroxyethyl)-3-methyltriaza-1-ene-2-oxide sodium salt was used instead of (Z)-3,3-diethyltriaza-1-ene-2-oxide sodium salt in the method, and reacted with compound 7 to obtain compound I2 in a yield of 70%.

[0049] The spectral data of compound I2 are as follows: 1H NMR (400 MHz, DMSO-d6) δ9.05 (d, J = 8.9 Hz,1H, ArH), 8.66 (d, J = 8.9 Hz, 1H, ArH), 8.58 (d, J = 7.2 Hz, 2H, ArH), 8.42(d, J = 15.1 Hz, 1H, ArH), 8.36 (d, J = 8.0 Hz, 1H, ArH), 8.33 – 8.29 (m, 1H,ArH), 8.21 – 8.11 (m, 2H, ArH), 7.95 (m, 1H, ArH), 7.82 (d, J = 8.3 Hz, 1H,ArH), 7.68 (m, 1H, ArH), 7.56 (m, 1H, CH), 7.39 (m, 1H, CH), 7.04 (m, 2H,ArH), 5.13 (d, J = 14.4 Hz, 1H, CH2), 4.75 (s, 2H, CH2), 4.54 (s, 3H, CH3), 4.47 (s, 3H, CH3), 4.25 (s, 3H, CH3), 3.47 (m, 2H, CH2), 3.17 (s, 3H, CH3), 2.74 (m, 2H, CH2).

[0050] The structural formula of compound I2 was determined as follows: Figure 1 As shown.

[0051] Example 3

[0052] Preparation of (E)-2-((4-((E)-(1,9-dimethyl-3-((E)-2-(1-methylquinoline-1-iodon-4-yl)vinyl)-9H-pyrido[3,4-b]indol-6-yl)diazeninyl)benzyl)oxy)-1-(pyrrolidine-1-yl)diazenin-1-oxide (compound I3):

[0053] Referring to the synthesis method of compound I1 in Example 1, (E)-2-onium salt-1-(pyrrolidine-1-yl)diazeline-1-oxide sodium salt was used instead of (Z)-3,3-diethyltriaza-1-ene-2-oxide sodium salt in the method, and reacted with compound 7 to obtain compound I3 with a yield of 77%.

[0054] The spectral data of compound I3 are as follows: 1H NMR (400 MHz, DMSO-d6) δ 9.08 – 9.01 (m, 1H,ArH), 8.66 (m, 1H, ArH), 8.57 (m, 2H, ArH), 8.44 – 8.38 (m, 1H, ArH), 8.37 –8.29 (m, 2H, ArH), 8.21 – 8.12 (m, 2H, ArH), 7.94 (m, 1H, ArH), 7.81 (m, 1H,ArH), 7.72 – 7.65 (m, 1H, CH,), 7.39 (m, 1H, ArH), 7.06 (m, 1H, CH), 6.84 (m,2H, ArH), 4.50 (s, 3H, CH3), 4.25 (s, 3H, CH3), 3.92 (s, 2H, CH2), 3.27 (m,4H, 2CH2), 3.17 (s, 3H, CH3), 1.93 (m, 4H, CH2).

[0055] The structural formula of compound I3 was determined as follows: Figure 1 As shown.

[0056] Example 4

[0057] Preparation of (E)-2-((4-((E)-(1,9-dimethyl-3-((E)-2-(1-methylquinoline-1-iodon-4-yl)vinyl)-9H-pyrido[3,4-b]indol-6-yl)diazeninyl)benzyl)oxy)-1-morpholinodiazenin-1-oxide (compound I4):

[0058] Referring to the synthesis method of compound I1 in Example 1, (E)-2-onium salt-1-morpholindiazene-1-oxide sodium salt was used instead of (Z)-3,3-diethyltriaza-1-ene-2-oxide sodium salt in the method, and reacted with compound 7 to obtain compound I4 with a yield of 72%.

[0059] The spectral data of compound I4 are as follows: 1H NMR (400 MHz, DMSO-d6) δ 8.93 (s, 1H, ArH), 8.69 (d, J = 15.8 Hz, 1H, ArH), 8.48 (d, J = 8.4 Hz, 1H, ArH), 8.42 (d, J =4.2 Hz, 2H, ArH), 8.30 (m, 2H, ArH), 8.26 – 8.22 (m, 1H, ArH), 8.16 (d, J =8.9 Hz, 1H, ArH), 8.01 (d, J = 15.8 Hz, 1H, ArH), 7.90 – 7.80 (m, 2H, ArH), 7.77 – 7.69 (m, 2H, ArH), 7.45 (m, 1H, CH), 7.19 (d, J = 15.7 Hz, 1H, CH), 4.85 (m, 2H, CH2), 4.27 (s, 3H, CH3), 3.51 (m, 4H, CH2), 3.20 (s, 3H, CH3), 3.08 (s, 3H, CH3), 2.78 (d, J = 7.5 Hz, 2H, CH2), 2.46 (s, 2H, CH2).

[0060] The structural formula of compound I4 was determined as follows: Figure 1 As shown.

[0061] Example 5

[0062] Preparation of (E)-2-((4-((E)-(1,9-dimethyl-3-((E)-2-(1-methylquinoline-1-iodon-4-yl)vinyl)-9H-pyrido[3,4-b]indol-6-yl)diazeninyl)benzyl)oxy)-1-(4-hydroxypiperidin-1-yl)diazenin-1-oxide (compound I5):

[0063] Referring to the synthesis method of compound I1 in Example 1, (E)-2-onium salt-1-(4-hydroxypiperidin-1-yl)diazeline-1-oxide sodium salt was used instead of (Z)-3,3-diethyltriaza-1-ene-2-oxide sodium salt in the method, and reacted with compound 7 to obtain compound I5 in a yield of 76%.

[0064] The spectral data of compound I5 are as follows: 1H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H, ArH),9.38 – 9.29 (m, 1H, ArH), 8.98 – 8.87 (m, 1H, ArH), 8.69 – 8.62 (m, 1H, ArH),8.61 – 8.46 (m, 3H, ArH), 8.40 – 8.22 (m, 3H, ArH), 8.09 (m, 1H, ArH), 7.83 –7.76 (m, 1H, ArH), 7.71 – 7.62 (m, 1H, CH), 7.37 (m, 1H, CH), 7.17 – 6.97 (m,2H, ArH), 5.01 (m, 2H, CH2), 4.29 – 4.17 (m, 3H, CH3), 3.97 (s, 3H, CH3), 3.17 (s, 3H, CH3), 2.88 (m, 1H, CH), 2.70 – 2.59 (m, 4H, CH2), 2.30-2.42 (m, 2H,CH2).

[0065] The structural formula of compound I5 was determined as follows: Figure 1 As shown.

[0066] Example 6 Fluorescence properties of the compounds of this invention in response to AzoR in hypoxic environments:

[0067] The mother liquors of the five compounds (I1-I5) obtained above were diluted to a concentration of 20 μM using a DMSO:PBS (5:95, v / v) mixture for detection. Changes in the fluorescence spectra in response to AzoR were detected by adding AzoR under an anaerobic environment. Using 480 nm as the excitation wavelength, fluorescence spectra of the compounds before and after the addition of AzoR were measured using a fluorescence spectrophotometer, collecting data from 500-800 nm.

[0068] like Figure 2 The fluorescence spectrum of the compound of the present invention in a DMSO:PBS (5:95, v / v) mixed solution is shown. Figure 2 a, 2b, 2c, 2d, and 2e are the fluorescence spectra of compounds I1-I5 in a DMSO:PBS (5:95, v / v) mixed solution under normoxic and hypoxic-AzoR conditions, respectively.

[0069] The results show that ( Figure 2The compounds of this invention exhibit very weak fluorescence signals under normoxic conditions. However, when incubated with AzoR in an anaerobic environment at a dose of 20 μM, the fluorescence intensity is significantly enhanced. This demonstrates the ability of the compounds of this invention to respond to AzoR in an anaerobic environment, undergoing spectral changes and generating near-infrared fluorescence signals amplified by 5 to 30 times.

[0070] Example 7

[0071] Selectivity and toxicity tests of the compounds of this invention against tumor cells:

[0072] The cytotoxicity of the compounds of this invention against human breast cancer MCF-7 and human colon cancer HT-29 cells under normoxic / hypoxic conditions (1% O2) was determined using the MTT assay. A flask of MCF-7 and HT29 cells in good exponential growth phase (Shanghai Institute of Cell Biology, China) was digested and prepared into 1×10⁶ cells / cells. 4 Cell suspensions of the present invention at concentrations of 1, 2, 5, 10, and 20 µM were seeded onto 96-well plates and incubated at 37°C for 12 hours. Then, different concentrations of the compound of the present invention were added, and the plates were cultured for another 72 hours under normoxic / hypoxic conditions (1% O2). Subsequently, 20 μL of MTT solution (5 mg / mL) was added to each well. -1 Four hours later, the MTT solution was discarded and 150 μL of DMSO was added. Finally, the absorbance at 490 nm was measured using a microplate reader.

[0073] Cell survival rate formula:

[0074] Survival rate (%) = (OD value of experimental group - OD value of blank control group) / (OD value of control group - OD value of blank control group) × 100%

[0075] The OD value refers to the optical density value. The OD value of the experimental group is the absorbance value of the treated cells, the OD value of the control group is the absorbance value of the untreated cells, and the OD value of the blank control group is the absorbance value of the culture medium.

[0076] in, Figure 3 a-3e represents the survival rate of HT-29 cells under normoxic and hypoxic conditions with different concentrations (1, 2, 5, 10, 20 µM) of compounds I1-I5. Figure 3 f-3j shows the survival rate of MCF-7 cells under normoxic and hypoxic conditions treated with different concentrations (1, 2, 5, 10, 20 µM) of compounds I1-I5.

[0077] The results show that ( Figure 3At different concentrations, all compounds of the present invention showed enhanced inhibitory effects on the growth of HT-29 colon cancer cells under hypoxic conditions compared to normoxic conditions. Notably, among the five compounds of the present invention, the cell inhibition rates against MCF-7 and HT-29 cells were significantly higher under hypoxic conditions than under normoxic conditions. Figure 3 (d,3i). This demonstrates that the compounds of the present invention have a good therapeutic effect on hypoxic tumors.

[0078] Example 8

[0079] The detection and testing of nitric oxide content in the compound of this invention:

[0080] Total nitric oxide was determined by detecting the nitrite content of five compounds (I1-I5) of this invention under normoxic or hypoxic-liver microsomal enzyme conditions using the classic Griess reagent.

[0081] in, Figure 4 The graph shows the nitrite concentration of the compounds of this invention under normal oxygen and hypoxic conditions (1% O2).

[0082] The results show that ( Figure 4 It is noteworthy that compound I4 exhibits a stronger ability to produce NO under hypoxic-hepatic microsomal enzyme conditions compared to normoxic conditions, and its content is also significantly higher than that of other compounds of this invention. The remaining compounds of this invention also demonstrate a stronger ability to produce NO under hypoxic conditions than under normoxic conditions, proving that the compounds of this invention can better kill tumors in the hypoxic tumor microenvironment, especially compound I4, which shows outstanding potential as a drug for treating tumors.

[0083] Example 9

[0084] In vivo fluorescence imaging and distribution testing of the compounds of this invention in nude mice:

[0085] Fluorescence signals at specified time points (2, 4, 8, 12, and 24 hours) were monitored in real time using a small animal imaging system to track the behavior of the compound of the present invention in vivo. Compound I4 (5 mg / kg) of the present invention was injected intravenously into nude mice carrying HepG2 orthotopic liver tumors. To determine the distribution profile of fluorescence within the specimens, the tumors and major organs were carefully excised after euthanasia of the nude mice for imaging analysis. To demonstrate the potential of the compound of the present invention as a valuable tool for improving tumor detection rates and facilitating image-guided surgical interventions, image-guided tumor surgery was performed to simulate the clinical surgical procedure.

[0086] in, Figure 5 a represents the in vivo fluorescence images of HepG2 tumor-bearing nude mice after tail vein injection of the compound of the present invention (5 mg / kg) at different times. Figure 5 b represents the average fluorescence intensity of the tumor area in a live nude mouse. Figure 5 c represents the surgical procedure of image-guided surgery mediated by the compounds of this invention. Figure 5 d represents representative in vitro fluorescence images of tumors and normal tissues. Figure 5 e represents the average fluorescence intensity of the tumor and major tissues.

[0087] The results showed that the fluorescence reached its peak intensity 12 hours after injection of compound I4 of the present invention, and was still detectable within 24 hours. Figure 5 (a, 5b). No fluorescent signal was detected in normal organs other than tumors. The approximately 22-fold fluorescence signal ratio between the tumor and the surrounding normal tissue indicates that compound I4 of the present invention accumulates or remains in tumors at a significantly higher rate. Figure 5 d, 5e). By using fluorescence images captured after injection of the compound of this invention, we were able to locate the liver tumor. Subsequently, the epidermis surrounding the tumor was surgically removed to expose the tumor tissue, which was then excised under the guidance of the fluorescence signal. Figure 5 c). This demonstrates that compound I4 of the present invention can specifically recognize liver tumors and effectively assist in guiding surgical resection of the tumor.

[0088] Example 10

[0089] Antitumor efficacy test of the compounds of this invention in nude mice:

[0090] To determine the antitumor effect of the compounds of this invention, we conducted pharmacodynamic studies on nude mice carrying HT29 tumors. The nude mice were divided into 4 groups: (a) control group ( Figure 6 (b) Capecitabine (clinically used as an adjuvant chemotherapy drug for colon cancer) Figure 6 (c) Compound I410 mg / kg of the present invention (Capecitabine group shown); Figure 6 (d) Compound I410 mg / kg (shown); (d) Compound I420 mg / kg of the present invention (shown); Figure 6 (As shown in the I420 mg / kg group). Control group mice were injected intratumorally with PBS solution, while the other three groups were injected intratumorally with the compound. Throughout the experiment, the body weight and tumor volume of all mice were monitored daily. Fifteen days after administration, all mice were euthanized, and their tumors were collected for further analysis, including measuring tumor volume and photographing.

[0091] in, Figure 6 Figure a shows the changes in tumor volume in nude mice over 15 days of treatment. Figure 6 b represents the tumor weight in a live nude mouse; Figure 6 c shows the weight changes of nude mice over 15 days of treatment; Figure 6 d is an anatomical tumor image 15 days after treatment.

[0092] The results showed that, compared with the control group, mice treated with the compound of the present invention exhibited a trend of inhibiting tumor growth, demonstrating superior efficacy compared with Capecitabine. Figure 6 (a, 6b, 6d). Furthermore, the body weight of the mice in all four groups did not fluctuate significantly, demonstrating that the compound of this invention has high safety for mice and does not affect their normal vital functions. Figure 6 c), demonstrating that the compounds of this invention have great potential for treating tumors.

Claims

1. A tumor hypoxia-responsive nitric oxide prodrug compound, characterized in that, The tumor hypoxia-responsive nitric oxide prodrug compound has the following structural formula: ， Wherein, R is selected from: , , , or Y is selected from Cl, Br, I, and PF6.

2. The tumor hypoxia-responsive nitric oxide prodrug compound according to claim 1, characterized in that, The tumor hypoxia-responsive nitric oxide prodrug compound is (Z)-1-((4-((E)-(1,9-dimethyl-3-((E)-2-(1-methylquinoline-1-iodon-4-yl)vinyl)-9H-pyrido[3,4-b]indol-6-yl)diazeninyl)benzyl)oxy)-3,3-diethyltriaza-1-ene-2-oxide, (E)-1-((4-((E)-) -(1,9-dimethyl-3-((E)-2-(1-methylquinoline-1-iodonium-4-yl)vinyl)-9H-pyrido[3,4-b]indol-6-yl)diazenyl)benzyl)oxy)-3-(2-hydroxyethyl)-3-methyltriazol-1-en-2-oxide, (E)-2-((4-((E)-(1,9-dimethyl-3-((E)-2-(1-methylquinoline-1-iodonium-4-yl)vinyl)-9H-pyrido[3,4-b]indol-6-yl)diazenyl)benzyl)oxy)-1-(pyrrolidine-1-yl)diazen-1-oxide, (E )-2-((4-((E)-(1,9-dimethyl-3-((E)-2-(1-methylquinoline-1-iodon-4-yl)vinyl)-9H-pyrido[3,4-b]indol-6-yl)diazenyl)benzyl)oxy)-1-morpholinodiazen-1-oxide, or (E)-2-((4-((E)-(1,9-dimethyl-3-((E)-2-(1-methylquinoline-1-iodon-4-yl)vinyl)-9H-pyrido[3,4-b]indol-6-yl)diazenyl)benzyl)oxy)-1-(4-hydroxypiperidin-1-yl)diazen-1-oxide).

3. The method for preparing the tumor hypoxia-responsive nitric oxide prodrug compound according to claim 1, characterized in that, The preparation method includes the following steps: 4-((E)-2-(6-((E)-(4-(bromomethyl)phenyl)diazeninyl)-1,9-dimethyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinolineonium salt (7) is reacted with azomonium oxide (RNa) to obtain the tumor hypoxia-responsive nitric oxide prodrug compound. The reaction route is shown below: 。 4. The preparation method according to claim 3, characterized in that, The bromo-4-((E)-2-(6-((E)-(4-(bromomethyl)phenyl)diazeninyl)-1,9-dimethyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinolineonium salt (7) was prepared by the following method: (E)-4-(2-(1,9-dimethyl-6-nitro-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinolineonium salt (4) was reduced with iron powder to obtain (E)-4-(2-(6-amino-1, 9-Dimethyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinolineonium salt (5) is then reacted with 4-nitrosobenzyl alcohol under acetic acid catalysis to generate 4-((E)-2-(6-((E)-(4-(hydroxymethyl)phenyl)diazeninyl)-1,9-dimethyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinolineonium salt (6). Finally, the bromo-4-((E)-2-(6-((E)-(4-(bromomethyl)phenyl)diazeninyl)-1,9-dimethyl-9H-pyrido[3,4-b]indol-3-yl)vinyl)-1-methylquinolineonium salt (7) is obtained by the Appel reaction under the action of CBr4 and PPh3. The reaction route is shown below: 。 5. A pharmaceutical composition, characterized in that, This includes the tumor hypoxia-responsive nitric oxide prodrug compound as described in claim 1 or 2, and a pharmaceutically acceptable carrier or excipient.

6. The use of the tumor hypoxia-responsive nitric oxide prodrug compound of claim 1 or 2 in the preparation of a fluorescent imaging reagent targeting liver tumors.

7. The use of the tumor hypoxia-responsive nitric oxide prodrug compound of claim 1 or 2 in the preparation of a targeted liver tumor localization drug.

8. The use of the tumor hypoxia-responsive nitric oxide prodrug compound of claim 1 or 2 in the preparation of tumor therapeutic drugs.

9. The use of the tumor hypoxia-responsive nitric oxide prodrug compound of claim 1 or 2 in the preparation of drugs for treating colon cancer and breast cancer.

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